4.1 Nutrition and Cell Chemistry Metabolism –The sum total of all chemical reactions that occur in a cell Catabolic reactions (catabolism) –Energy-releasing metabolic reactions Anabolic reactions (anabolism) –Energy-requiring metabolic reactions Most knowledge of microbial metabolism is based on study of laboratory cultures © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Nutrients –Supply of monomers (or precursors of) required by cells for growth Macronutrients –Nutrients required in large amounts Micronutrients –Nutrients required in trace amount © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Carbon –Required by all cells –Typical bacterial cell ~50% carbon (by dry weight) –Major element in all classes of macromolecules –Heterotrophs use organic carbon –Autotrophs use inorganic carbon © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Nitrogen –Typical bacterial cell ~12% nitrogen (by dry weight) –Key element in proteins, nucleic acids, and many more cell constituents © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Other Macronutrients –Phosphorus (P) Synthesis of nucleic acids and phospholipids –Sulfur (S) Sulfur-containing amino acids (cysteine and methionine) Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme A –Potassium (K) Required by enzymes for activity © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Other Macronutrients (cont’d) –Magnesium (Mg) Stabilizes ribosomes, membranes, and nucleic acids Also required for many enzymes –Calcium (Ca) Helps stabilize cell walls in microbes Plays key role in heat stability of endospores –Sodium (Na) Required by some microbes (e.g., marine microbes) © 2012 Pearson Education, Inc.

4.1 Nutrition and Cell Chemistry Iron –Key component of cytochromes and FeS proteins involved in electron transport –Under anoxic conditions, generally ferrous (Fe 2+ ) form; soluble –Under oxic conditions: generally ferric (Fe 3+ ) form; exists as insoluble minerals –Cells produce siderophores (iron-binding agents) to obtain iron from insoluble mineral form (Figure 4.2) © 2012 Pearson Education, Inc.

4.2 Culture Media Culture Media –Nutrient solutions used to grow microbes in the laboratory Two broad classes –Defined media: precise chemical composition is known –Complex media: composed of digests of chemically undefined substances (e.g., yeast and meat extracts) © 2012 Pearson Education, Inc.

4.2 Culture Media Selective Media –Contains compounds that selectively inhibit growth of some microbes but not others Differential Media –Contains an indicator, usually a dye, that detects particular chemical reactions occurring during growth © 2012 Pearson Education, Inc.

4.5 Catalysis and Enzymes Enzymes –Biological catalysts –Typically proteins (some RNAs) –Highly specific –Generally larger than substrate –Typically rely on weak bonds Examples: hydrogen bonds, van der Waals forces, hydrophobic interactions –Active site: region of enzyme that binds substrate © 2012 Pearson Education, Inc.

Figure 4.7 Substrate Products Active site Free lysozyme Enzyme-substrate complex © 2012 Pearson Education, Inc.

III. Oxidation–Reduction and Energy- Rich Compounds 4.6Electron Donors and Electron Acceptors 4.7Energy-Rich Compounds and Energy Storage © 2012 Pearson Education, Inc.

4.6 Electron Donors and Electron Acceptors Energy from oxidation–reduction (redox) reactions is used in synthesis of energy-rich compounds (e.g., ATP) Redox reactions occur in pairs (two half reactions; Figure 4.8) Electron donor: the substance oxidized in a redox reaction Electron acceptor: the substance reduced in a redox reaction © 2012 Pearson Education, Inc.

Essentials of Catabolism 4.8 Glycolysis 4.9 Respiration and Electron Carriers 4.10 The Proton Motive Force 4.11 The Citric Acid Cycle 4.12 Catabolic Diversity © 2012 Pearson Education, Inc.

4.8 Glycolysis Two reaction series are linked to energy conservation in chemoorganotrophs: fermentation and respiration (Figure 4.13) Differ in mechanism of ATP synthesis –Fermentation: substrate-level phosphorylation; ATP directly synthesized from an energy-rich intermediate –Respiration: oxidative phosphorylation; ATP produced from proton motive force formed by transport of electrons © 2012 Pearson Education, Inc.

4.8 Glycolysis Fermented substance is both an electron donor and an electron acceptor Glycolysis (Embden-Meyerhof pathway): a common pathway for catabolism of glucose (Figure 4.14) –Anaerobic process –Three stages © 2012 Pearson Education, Inc.

Figure 4.14 Stage I Stage II Stage III Glucose Pyruvate 2 Pyruvate 2 lactate 2 ethanol  2 CO 2 Energetics Yeast Lactic acid bacteria Intermediates Glucose 6-P Fructose 6-P Fructose 1,6-P Dihydroxyacetone-P Glyceraldehyde-3-P 1,3-Bisphosphoglycerate 3-P-Glycerate 2-P-Glycerate Phosphoenolpyruvate Enzymes Hexokinase Isomerase Phosphofructokinase Aldolase Triosephosphate isomerase Glyceraldehyde-3-P dehydrogenase Phosphoglycerokinase Phosphoglyceromutase Enolase Pyruvate kinase Lactate dehydrogenase Pyruvate decarboxylase Alcohol dehydrogenase © 2012 Pearson Education, Inc.

4.8 Glycolysis Glycolysis –Glucose consumed –Two ATPs produced –Fermentation products generated Some harnessed by humans for consumption © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers Aerobic Respiration –Oxidation using O 2 as the terminal electron acceptor –Higher ATP yield than fermentations ATP produced at the expense of the proton motive force, which is generated by electron transport © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers Electron Transport Systems –Membrane associated –Mediate transfer of electrons –Conserve some of the energy released during transfer and use it to synthesize ATP –Many oxidation–reduction enzymes are involved in electron transport (e.g., NADH dehydrogenases, flavoproteins, iron–sulfur proteins, cytochromes) © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers NADH dehydrogenases: proteins bound to inside surface of cytoplasmic membrane; active site binds NADH and accepts 2 electrons and 2 protons that are passed to flavoproteins Flavoproteins: contains flavin prosthetic group (e.g., FMN, FAD) that accepts 2 electrons and 2 protons but only donates the electrons to the next protein in the chain (Figure 4.15) © 2012 Pearson Education, Inc.

Figure 4.15 Isoalloxazine ring Ribitol Oxidized Reduced © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers Cytochromes –Proteins that contain heme prosthetic groups (Figure 4.16) –Accept and donate a single electron via the iron atom in heme © 2012 Pearson Education, Inc.

Figure 4.16 Porphyrin ring Pyrrole Heme (a porphyrin) Histidine-N Cysteine-S Amino acid S-Cysteine N-Histidine Protein Cytochrome © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers Iron–Sulfur Proteins –Contain clusters of iron and sulfur (Figure 4.17) Example: ferredoxin –Reduction potentials vary depending on number and position of Fe and S atoms –Carry electrons © 2012 Pearson Education, Inc.

Figure 4.17 Cysteine © 2012 Pearson Education, Inc.

4.9 Respiration and Electron Carriers Quinones –Hydrophobic non-protein-containing molecules that participate in electron transport (Figure 4.18) –Accept electrons and protons but pass along electrons only © 2012 Pearson Education, Inc.

4.10 The Proton Motive Force Electron transport system oriented in cytoplasmic membrane so that electrons are separated from protons (Figure 4.19) Electron carriers arranged in membrane in order of their reduction potential The final carrier in the chain donates the electrons and protons to the terminal electron acceptor © 2012 Pearson Education, Inc.

Figure 4.19 CYTOPLASM ENVIRONMENT Complex II Succinate Fumarate Complex I Complex III Complex IV Q cycle   0.1  0.36  0.39 E 0 (V) © 2012 Pearson Education, Inc.

4.10 The Proton Motive Force During electron transfer, several protons are released on outside of the membrane –Protons originate from NADH and the dissociation of water Results in generation of pH gradient and an electrochemical potential across the membrane (the proton motive force) –The inside becomes electrically negative and alkaline –The outside becomes electrically positive and acidic © 2012 Pearson Education, Inc.

4.10 The Proton Motive Force Complex I (NADH:quinone oxidoreductase) –NADH donates e  to FAD –FADH donates e  to quinone Complex II (succinate dehydrogenase complex) –Bypasses Complex I –Feeds e  and H + from FADH directly to quinone pool © 2012 Pearson Education, Inc.

4.10 The Proton Motive Force Complex III (cytochrome bc 1 complex) –Transfers e  from quinones to cytochrome c –Cytochrome c shuttles e  to cytochromes a and a 3 Complex IV (cytochromes a and a 3 ) –Terminal oxidase; reduces O 2 to H 2 O © 2012 Pearson Education, Inc.

4.10 The Proton Motive Force ATP synthase (ATPase): complex that converts proton motive force into ATP; two components (Figure 4.20) –F 1 : multiprotein extramembrane complex, faces cytoplasm –F o : proton-conducting intramembrane channel –Reversible; dissipates proton motive force © 2012 Pearson Education, Inc.

Figure 4.20 Membrane Out In F1F1 F1F1 FoFo FoFo c 12 b2b2 b2b2 a a c              © 2012 Pearson Education, Inc.

4.11 The Citric Acid Cycle Citric acid cycle (CAC): pathway through which pyruvate is completely oxidized to CO 2 (Figure 4.21a) –Initial steps (glucose to pyruvate) same as glycolysis –Per glucose molecule, 6 CO 2 molecules released and NADH and FADH generated –Plays a key role in catabolism and biosynthesis Energetics advantage to aerobic respiration (Figure 4.21b) © 2012 Pearson Education, Inc.

Figure 4.21a Pyruvate  (three carbons) Acetyl-CoA Oxalacetate 2  Malate 2  Fumarate 2  Succinate 2  Succinyl-CoA Citrate 3  Aconitate 3  Isocitrate 3   -Ketoglutarate 2  C2C2 C4C4 C5C5 C6C6 © 2012 Pearson Education, Inc.

Figure 4.21b Energetics Balance Sheet for Aerobic Respiration © 2012 Pearson Education, Inc.

4.11 The Citric Acid Cycle The citric acid cycle generates many compounds available for biosynthetic purposes –  -Ketoglutarate and oxalacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose –Succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds –Acetyl-CoA: necessary for fatty acid biosynthesis © 2012 Pearson Education, Inc.

4.12 Catabolic Diversity Microorganisms demonstrate a wide range of mechanisms for generating energy (Figure 4.22) – Fermentation – Aerobic respiration – Anaerobic respiration – Chemolithotrophy – Phototrophy © 2012 Pearson Education, Inc.

Figure 4.22 Fermentation Carbon flow Organic compound Carbon flow in respirations Electron transport/ generation of pmf Aerobic respiration Biosynthesis Organic compound Electron acceptors Anaerobic respiration Chemoorganotrophy Chemolithotrophy Phototrophy Electron transport/ generation of pmf Anaerobic respiration Electron acceptors Aerobic respiration Light Photoheterotrophy Photoautotrophy Electron transport Generation of pmf and reducing power e  donor Chemotrophs Phototrophs © 2012 Pearson Education, Inc.

4.12 Catabolic Diversity Anaerobic Respiration –The use of electron acceptors other than oxygen Examples include nitrate (NO 3  ), ferric iron (Fe 3+ ), sulfate (SO 4 2  ), carbonate (CO 3 2  ), certain organic compounds –Less energy released compared to aerobic respiration –Dependent on electron transport, generation of a proton motive force, and ATPase activity © 2012 Pearson Education, Inc.

4.12 Catabolic Diversity Chemolithotrophy –Uses inorganic chemicals as electron donors Examples include hydrogen sulfide (H 2 S), hydrogen gas (H 2 ), ferrous iron (Fe 2+ ), ammonia (NH 3 ) –Typically aerobic –Begins with oxidation of inorganic electron donor –Uses electron transport chain and proton motive force –Autotrophic; uses CO 2 as carbon source © 2012 Pearson Education, Inc.

4.12 Catabolic Diversity Phototrophy: uses light as energy source –Photophosphorylation: light-mediated ATP synthesis –Photoautotrophs: use ATP for assimilation of CO 2 for biosynthesis –Photoheterotrophs: use ATP for assimilation of organic carbon for biosynthesis © 2012 Pearson Education, Inc.